Biosensor for defecting chemical agents

ABSTRACT

A biosensor for the identification of olfactants, including chemical and biological agents, prefumery, explosives and pharmaceuticals. The biosensors comprise robust eukaryotic cells, such as yeast, into which at least one exogenous olfactory signaling pathway has been genetically integrated to detect molecules of interest.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the priority of copending U.S. ProvisionalApplication Ser. No. 60/267,223 filed Feb. 8, 2001, the entiredisclosure of which is herein incorporated by reference.

REFERENCE TO GOVERNMENT GRANT

The invention described herein was supported in part by the Departmentof Advance Research Agency (DARPA) of the Department of Defense, undergrant no. N66001-00-C-8050. The government has certain rights in thisinvention.

FIELD OF THE INVENTION

This invention pertains to the field of molecular biosensors, inparticular biosensors containing cloned olfactory receptors.

BACKGROUND OF THE INVENTION

The olfactory receptors are a class of G-protein coupled receptors(GPCR) found in the membranes of cells in olfactory or other sensingorgans. The olfactory receptor proteins are considered the largestsub-family of GPCR; it has been estimated that human olfactoryepithelium contains approximately 2000-3000 distinct olfactoryreceptors, whereas insects such as Drosophila have 100-200 suchreceptors. It is believed that any given olfactory or sensory cellcontains one or a few types of olfactory receptors.

Olfactory receptor proteins have a distinctive structure of sevenhydrophobic segments that span the cell membrane (trans-membrane domainsI-VII), separated by hydrophilic segments which project into the intra-or extra-cellular space. Transmembrane domains II through VII comprise ahypervariable segment which defines the ligand specificity of thereceptor. This hypervariable segment is flanked by hydrophobic consensussequences. Furthermore, it is known that the N-terminal segment of theolfactory receptor is involved in receptor stability and the C-terminalsegment is involved in G-protein coupling and activation. The structuralorganization of a typical olfactory receptor is given in FIG. 1.

The basic olfactory signaling unit consists of an olfactory receptor, asignal transducer (e.g., G-protein), an effector (e.g., adenylylcyclase), second messengers (e.g., cAMP), and a gated channel (e.g., acalcium channel) as depicted in FIG. 2. Olfactory receptor signaling isnot, however, limited to the G-protein-adenylyl cyclase-cAMP pathway;there is evidence of olfactory receptor signaling via G-proteinactivation of phosphoinositidase C, with subsequent production ofinositol 1,4,5-triphosphate and 1,2-diacylglycerol second messengers.

The method of chemical detection by olfactory receptors is conservedamong species. Upon activation by an olfactant, an olfactory receptorinitiates a cellular signaling cascade that results in the influx ofcalcium ions, which in turn leads to a depolarization of a connectedsensory neuron. The time from receptor activation by binding of ligandto calcium influx is typically a few milliseconds.

The olfactory receptors are highly sensitive and selective; for example,they can detect femtomolar concentrations (10⁻¹³ M) of a specificchemical molecule and distinguish between two molecules differing in asingle hydrogen atom.

The great variety, exquisite specificity and sensitivity, andfast-acting properties of the olfactory receptors make them idealcomponents of a biosensor. As its name implies, a biosensor is adetector that has a biological sensory component, such as a receptorprotein or nucleic acid. Biosensors offer the advantages of higherresolution and the possibility of real-time monitoring of environmentsover conventional analytical techniques.

Also, because a biosensor can be constructed at the cellular ormolecular level, many biosensors capable of detecting one or moresubstances can be contained in a very small area. Modem molecularbiology and genetic techniques also allow a large number of diversebiological sensors to be generated quickly and cheaply.

However, several characteristics of typical eukaryotic expressionsystems, and of naturally occurring olfactory receptors, have preventedthe production of a robust biological sensor that can be easily adaptedto detect diverse substances. In particular, cloning and expression ofolfactory receptors has been inhibited by the inability of many hostcells to properly process and transport the receptors to the cellmembrane.

Even if the olfactory receptors are properly positioned by the hostcell, they are often not coupled to an appropriate second-messengersystem. Coupling of olfactory receptors to their effectors appears to behighly specific, and endogenous G-proteins in heterologous host cellsmay not efficiently transduce and amplify the olfactory receptor'ssignal upon ligand binding. To overcome this shortcoming, host cellssuch as melanophores expressing large numbers of endogenous G_(alpha)subunits (thus increasing the probability of an effective coupling) areoften used. See TS McClintock et al. (1997), Molec. Brain Res. 48:270-278. Alternatively, G_(alpha) subunits which couple to a variety ofreceptors, such as the G_(α15,16) subunits, are co-transfected into thehost cell with the olfactory receptor. See Offermans and Simon (1995),J. Biol. Chem. 270(25): 15175-15180. However, such techniques do notreliably couple every olfactory receptor to a second-messenger.

Moreover, eukaryotic expression systems typically consist of culturedmammalian cells. Such systems are not robust, and require specializedhandling under laboratory culture conditions to be viable.

The increasing threat of chemical and biological warfare agents in warand terrorist acts requires a biosensor that can operate in urban areasand battlefields alike under field conditions. The biosensor must beportable, rugged, sensitive, and reliable. What is needed, therefore, isa biosensor that can operate under the environmental conditions expectedto be encountered outside the laboratory, and which reliably andreproducibly detects olfactants of interest.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the structural organization of a typical olfactoryreceptor.

FIG. 2 shows the basic olfactory signaling unit.

FIG. 3 shows the primary and secondary structure of a chimeric olfactoryreceptor.

FIG. 4A shows the sensitivity of biosensor WIF-1α-RI7 by plotting theratio of WIF-1α-RI7 GFP fluorescence values over pre-biosensor WIF-1α(control) GFP fluorescence values for different olfactants at varyingconcentrations (25, 50, 75, and 100 nM). FIG. 4B shows the specificityof biosensor WIF-1α-RI7 for different olfactants by plotting the ratioof WIF-1α-RI7 GFP fluorescence values over pre-biosensor WIF-1α(control) GFP fluorescence values for different olfactants at a singleconcentration (25 nM).

FIG. 5A is a photograph of a 1% agarose gel showing detection of a 984bp RI7 PCR fragment from control and WIF-1α cells. FIG. 5B is Westernblot showing detection of CREBP and Gαolf protein in control and WIF-1αcells.

FIG. 6 shows black-and-white representations of laser confocalmicrographs showing expression of GFP in WIF-1α-RI7 cells that were A)not exposed to any olfactant (None) or exposed to B) 100 nmoles ofhexaldehyde (6-CHO); C) 100 nmoles heptaldehyde (7-CHO); or D) 100nmoles of octaldehyde (8-CHO).

DEFINITIONS

The following definitions are provided to aid in the understanding ofthe invention:

As used herein, the term “chimeric protein” refers to two or morenucleotide sequences obtained from different genes that have been clonedtogether and that encode a single polypeptide segment. Chimeric proteinsare also referred to as “hybrid proteins.” As used herein, the term“chimeric protein” refers to coding segments that are obtained fromdifferent species of organisms, as well as coding segments that areobtained from the same species of organisms.

As used herein, “conservative amino acid substitutions” aresubstitutions with an amino acid which has a related side chain R. Aminoacids are typically classified into seven groups on the basis of theside chain R: (1) aliphatic side chains, (2) side chains containing ahydroxylic (OH) group, (3) side chains containing sulfur atoms, (4) sidechains containing an acidic or amide group, (5) side chains containing abasic group, (6) side chains containing an aromatic ring, and (7)proline, an imino acid in which the side chain is fused to the aminogroup.

As used herein, or “couples” or “coupling” refers to the physical and/orfunctional connection between two components of a cellular pathway orsystem. For example, the C-terminal segment of an olfactory receptorcouples the receptor to the G-protein, allowing the activation of theG-protein upon binding of ligand to the receptor. The olfactory receptorC-terminal segment is an example of a “coupling segment,” which is asegment of a protein or other cellular component that couples to anothercomponent.

As used herein, the term “cloning cassette” refers to a nucleic acidsequence that contains multiple cloning sites (i.e., “restrictionsites,” “MCS,” or “polylinker”). It is intended that the term encompassDNA that contains unique, as well as non-unique restriction sites.

As used herein, “encoding” or “encoded” with respect to a specifiednucleic acid means the genetic information contained within the nucleicacid may be translated into a specified protein. A nucleic acid encodinga protein may comprise non-translated sequences (e.g., introns) withintranslated regions of the nucleic acid, or may lack such interveningnon-translated sequences (e.g., as in cDNA). The information by which aprotein is encoded is specified by the use of codons. Typically, theamino acid sequence is encoded by the nucleic acid using the “universal”genetic code. However, variants of the universal code, such as arepresent in some plant, animal, and fungal mitochondria, the bacteriumMycoplasma capricolum (Proc. Natl. Acad. Sci. (USA), 82: 2306-2309(1985)), or the ciliate Macronucleus, may be used when the nucleic acidis expressed using these organisms.

As used herein, the terms “express” or “expression” mean the productionof a functional protein product from the genetic information containedwithin a nucleic acid sequence.

As used herein, “expression cassettes” are DNA constructs that include,5′ to 3′ in the direction of transcription, a promoter, a DNA sequenceoperatively associated with the promoter, and, optionally, a terminationsequence including a stop signal for RNA polymerase and apolyadenylation signal for polyadenylase. All of these regulatoryregions should be capable of operating in the cell to be transfected.Suitable termination signals for a given DNA construct will be apparentto those of skill in the art. The term “operatively associated,” as usedherein, refers to the relationship between DNA sequences in a single DNAmolecule which are associated so that the function of one sequence isaffected by the other. Thus, a promoter is operatively associated with aDNA when it is capable of affecting the transcription of that DNA (i.e.,the DNA is under the transcriptional control of the promoter). Thepromoter is upstream (5′) from the DNA, which is in turn said to bedownstream (3′) from the promoter.

An expression cassette may be provided in a vector construct which alsohas at least one replication system. For convenience, it is common toemploy a replication system functional in Escherichia coli, such asCo1E1, pSC101, pACYC184, the pUC plasmids, or the like. In this manner,at each stage after each manipulation, the resulting construct may becloned, sequenced, and the correctness of the manipulation determined.In addition, or in place of an E. coli replication system, a broad hostrange replication system may be employed, such as the replicationsystems of the P-1 incompatibility plasmids, e.g., pRK290. In additionto the replication system, there will frequently be at least one markerpresent in an expression cassette, which may be useful in one or morehosts, or different markers for individual hosts. That is, one markermay be employed for selection in a prokaryotic host, while anothermarker may be employed for selection in a eukaryotic host. The markersmay comprise protection against a biocide, such as antibiotics, toxins,heavy metals, or the like; may provide complementation, by impartingprototrophy to an auxotrophic host; or may provide a visible phenotypethrough the production of a novel compound in the host.

Various fragments comprising the various constructs, expressioncassettes, markers, and the like may be introduced consecutively byrestriction enzyme cleavage of an appropriate replication system, andinsertion of the particular construct or fragment into the availablesite. After ligation and cloning the DNA construct may be isolated forfurther manipulation. All of these techniques are know to those skilledin the art, and are amply illustrated in the literature, as exemplifiedby J. Sambrook et al., Molecular Cloning, A Laboratory Manual (2d Ed.1989) (Cold Spring Harbor Laboratory).

As used herein, “exogenous” refers to a substance (e.g., protein, DNA orRNA) or system (e.g., a second-messenger cell signaling pathway) whichhas been introduced into a cell or the cell's ancestor through theefforts of humans. Such substances or systems may be a copy of somethingnaturally found in the cell being transformed, or a sequence which isnot naturally found in the cell being transformed, or fragments thereof.

As used herein, the term “gene” refers to a DNA sequence thatincorporates (1) upstream (5′) regulatory signals including a promoter,(2) a coding segment specifying the product, protein or RNA of the gene,(3) downstream (3′) regions including transcription termination andpolyadenylation signals and (4) associated sequences required forefficient and specific expression.

As used herein, “Green Fluorescent Protein” or “GFP” refers to thevarious naturally occurring forms of GFP which can be isolated fromnatural sources, as well as artificially modified GFPs which retain thefluorescent abilities of native GFP.

As used herein, the term “heterologous” means the substances, systemsand/or cells being referred to do not have the same origin; or, if fromthe same origin, are substantially modified from their native form bydeliberate human intervention.

As used herein, “nucleic acid” includes reference to adeoxyribonucleotide or ribonucleotide polymer in either single- ordouble-stranded form, and unless otherwise limited, encompasses knownanalogues having the essential nature of natural nucleotides in thatthey hybridize to single-stranded nucleic acids in a manner similar tonaturally occurring nucleotides (e.g., peptide nucleic acids).

As used herein, “olfaction” or “olfactory reception” means the detectionof compounds by an olfactory receptor coupled to a cell signalingpathway. The compound detected is termed an “olfactant” and may beair-borne (i.e., volatile) or in solution.

The term “promoter” refers to a region of a DNA sequence thatincorporates the necessary signals for the efficient expression of acoding sequence. This may include sequences to which an RNA polymerasebinds but is not limited to such sequences and may include regions towhich other regulatory proteins bind together with regions involved inthe control of protein translation and may include coding sequences.Suitable promoters will be apparent to those skilled in the art, andwill vary depending upon the cell in which the DNA is to be expressed.Both inducible and constitutive promoters are contemplated for use inthe present invention.

As used herein, “a regulatory element” from a gene is the DNA sequencewhich is necessary for the expression of the gene, such as a promoter orresponsive element. In this invention, the term “operatively linked”means that a regulatory element can direct the expression of a linkedDNA sequence.

As used herein, a “robust cell” is a cell which is viable outside ofcontrolled laboratory culture conditions and can perform the biochemicalreactions necessary for olfactory detection in the field and, ifnecessary, under environmental extremes. Robust cells include thosecomprising a living organism that can be taken into the field, forexample nematode, fish or insect species.

As used herein, the terms “transfection” or “transfected” includereference to the incorporation of a nucleic acid into a eukaryotic orprokaryotic cell where the nucleic acid may be incorporated into thegenome of the cell (e.g., chromosome, plasmid, plastid or mitochondrialDNA), converted into an autonomous replicon, or transiently expressed(e.g., transfected mRNA). A cell has been “transfected” by exogenous orheterologous DNA when such DNA has been introduced inside the cell.

As used herein, “vector” includes reference to a nucleic acid used intransfection of a host cell and into which can be inserted apolynucleotide. Vectors are often replicons. Expression vectors permittranscription of a nucleic acid inserted therein.

As used herein, the term “host cell” refers to any cell transfected witha nucleic acid for expressing a protein product, or that cell's progeny.A transfected cell or its progeny is considered a “host cell” as long asthe nucleic acid for expressing a protein product is present in thecell, whether the nucleic acid is integrated into the cell's genome orexists as an extrachromosomal element. As used herein, a “host mastercell” refers to a host cell transfected to express at least onesignaling pathway. It is understood that a variety of master host cellsmay be constructed using different signaling pathways. Host master cellsare used to construct “pre-biosensors,” which are master host cellstransfected to express at least one signal reporters. It is understoodthat a variety of pre-biosensor cells may be constructed from a givenmaster host cell by transfection with different signal reporter DNA. Thepre-biosensor cells are used to construct the biosensors of theinvention, by transfection of pre-biosensor cells with at least oneexpression vector comprising a chimeric olfactory receptor protein.Master host cells, pre-biosensor cells, and biosensors, and methods ofmaking and using them are described more fully below.

As used herein, a “signaling pathway” refers to a system of cellularcomponents that transduces the signal produced from the binding of aligand to a membrane-bound receptor into a biological effect. Forexample, a signaling pathway may comprise an intracellular G-protein,its associated effector protein and a gated ion channel in the cellmembrane. The effector protein may comprise an enzyme, such as aphosphorylase or cyclase, and may be integral with the G-protein.Typically, the G-protein is activated by the binding of ligand to areceptor. The activated G-protein in turn activates the effector toproduce a second messenger. The second messenger produces somebiological effect in the cell, such as upregulating gene transcriptionor opening a gated ion channel. The production of a second messenger isalso considered a biological effect. As used herein, the signalingpathway does not include the membrane-bound receptor; the signalingpathway plus receptor is termed a “signaling unit.” Signaling pathwaysare described in more detail below.

As used herein, a “signal reporter” refers to one or more substances incell that are responsive to a biological effect produced by theactivation of the signaling pathway, for example changes in theintracellular concentration of an ion or second messenger. The signalreporter may comprise an indicator that produces a detectable phenomenon(e.g., fluorescence, change in electric potential of the cell membrane,etc.), or may be an integrated system of components that combine toproduce a detectable phenomenon. Signal reporters are described in moredetail below.

Abbreviations

The following is a list of abbreviations used in the specification:

-   Ca⁺⁺—calcium ion-   cAMP—cyclic adenosine monophosphate-   CREB—cAMP-responsive element binding protein-   CRE—cAMP responsive element-   DNA—deoxyribonucleic acid-   EDTA—ethylene diamine tetraacetic acid-   FURA-2—1-[6-Amino-2-(5-carboxy-2-oxazolyl)-5-benzofuranyloxy]-2-(2-amino-5-methylphenoxy)ethane-N,N,N′,N′-tetraacetic    acid, penta-potassium salt-   GFP—green fluorescent protein-   Golf—olfactory receptor G-protein-   GPCR—G protein coupled receptor-   LiAc—lithium acetate-   mM—millimolar-   mm—millimeter-   mg—milligram-   ng—nanogram-   nm—nanometers-   PCR—polymerase chain reaction-   RT-PCR—reverse-transcription polymerase chain reaction-   RNA—ribonucleic acid-   TE—Tris/EDTA-   x-gal-5-bromo-4 chloro-3-indolyl-b-D-galactopyranoside-   Amino Acids—The standard one- and three-letter amino acid    abbreviations are used, as set forth in the following schedule:

A Alanine Ala C Cysteine Cys D Aspartic Acid Asp E Glutamic Acid Glu FPhenylalanine Phe G Glycine Gly H Histidine His I Isoleucine Ile KLysine Lys L Leucine Leu M Methionine Met N Asparagine Asn P Proline ProQ Glutamine Gln R Arginine Arg S Serine Ser T Threonine Thr V Valine ValW Tryptophan Trp Y Tyrosine TyrThe prefix “L-” preceding an amino acid abbreviation (e.g., “L-Trp”)denotes the biologically active levorotatory isomer of the amino acid.

SUMMARY OF THE INVENTION

The present invention provides a biosensor comprising one or more hostcells expressing at least one chimeric olfactory receptor protein forbinding olfactant; at least one exogenous signaling pathway coupled tothe at least one chimeric olfactory receptor for transducing a signalproduced by the chimeric olfactory receptor upon olfactant binding; andat least one signal reporter coupled to the signaling pathway forproducing a detectable phenomenon upon transduction of the olfactantbinding signal by the signaling pathway. The chimeric olfactory receptorprotein expressed by the biosensor comprises an olfactory receptorhypervariable segment which contains at least one olfactant bindingsite, a processing/transport segment which directs the processing andtransport of the chimeric receptor in the host cell; and a couplingsegment which couples the chimeric receptor to an exogenous signalingpathway in the host cell. In preferred embodiments, the expressedchimeric receptor comprises an N-terminal processing/transport signalsegment and a C-terminal G-protein receptor coupling segment flanking anolfactory receptor hypervariable segment.

The invention also provides a library of biosensors comprising aplurality of biosensors with differing olfactant specificities.

The invention also provides host master cells, which are host cellstransfected to express at least one exogenous signaling pathway.

The invention also provides a pre-biosensor comprising a host mastercell of the invention further transfected to express at least one signalreporter.

The invention also provides a basic expression vector comprising acloning cassette and nucleotide sequences encoding aprocessing/transport signal segment and a G-protein receptor couplingsegment operative in an olfactory receptor protein. A nucleotidesequence encoding an olfactory receptor hypervariable segment may beinserted into the cloning cassette to produce an expression vector thatcan express a chimeric olfactory receptor protein. In preferredembodiments, the nucleotide sequences encoding an olfactory receptorhypervariable segment a coupling segment flank the cloning cassette.

The invention also provides an expression vector comprising nucleotidesequences encoding a chimeric olfactory receptor protein, wherein thereceptor protein comprises (i) an olfactory receptor hypervariablesegment which contains at least one olfactant binding site; (ii) aprocessing/transport segment which directs the processing and transportof the chimeric receptor in the host cell; and (iii) a coupling segmentwhich couples the chimeric receptor to an exogenous signaling pathway inthe host cell. In preferred embodiments, the chimeric receptor proteincomprises (i) an olfactory receptor hypervariable segment; (ii) aprocessing/transport signal segment N-terminal to the olfactory receptorhypervariable segment; and (iii) a G-protein receptor coupling segmentC-terminal to the olfactory receptor hypervariable segment.

The invention also provides an expression vector library comprisingexpression vectors which contain different olfactory receptorhypervariable segments. Such a library may be used to construct alibrary of biosensors with differing olfactant specificities.

The invention also provides a method of producing a biosensor,comprising transfecting one or more host cells to express (i) at leastone chimeric olfactory receptor protein for binding olfactant; (ii) atleast one exogenous signaling pathway coupled to the at least onechimeric olfactory receptor for transducing a signal produced by thechimeric olfactory receptor upon olfactant binding; and (iii) at leastone signal reporter coupled to the signaling pathway for producing adetectable phenomenon upon transduction of the olfactant binding signalby the signaling pathway.

The invention further provides a method of identifying biosensors whichcan detect an olfactant, comprising the steps of providing at least onebiosensor of the invention, wherein detection of the olfactant by thebiosensor generates a detectable phenomenon from the signal reportercontacting the one or more biosensor with the olfactant; contacting theat least one biosensor with an olfactant, and observing whether thedetectable phenomenon is produced from the signal reporter.

The invention further provides a method for detecting an olfactant in asample, comprising the steps of providing one or more biosensors of theinvention capable of detecting an olfactant, wherein detection of theolfactant by the biosensor generates a detectable phenomenon from thesignal reporter; contacting the one or more biosensors with a samplesuspected of containing the olfactant; and observing whether thedetectable phenomenon is produced from the biosensor signal reporter.

The invention further provides an apparatus capable of detecting thedetectable phenomenon produced by detection of an olfactant by abiosensor of the invention. The apparatus comprises a measurement toolfor measuring the detectable phenomenon, means for controlling themeasurement tool (e.g., a computer controller) and one or morebiosensors of the invention preferably comprising an array or biochip.

The invention further provides kits comprising various combinations ofthe components for constructing the biosensors of the invention.

The invention further provides arrays and portable containers comprisingbiosensors of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The present biosensors are designed for use in the field, underpotentially harsh or changeable environmental conditions. As such, thebiosensors are constructed from cells able to survive outside of thecontrolled environment of the laboratory for more than a limited time.

The present biosensors are therefore preferably constructed from robustcells. Robust cells useful in the invention may be obtained from anyprokaryotic or eukaryotic organism which is adapted to live in a harshor changeable environment. For example, some species of bacteria canexist in extremes of temperature (e.g., Thermus aquaticus and otherthermophilic bacteria) in high-salt (e.g., halobacteria) or low-oxygenenvironments, under conditions of acidity or basicity, in high pressure,or combinations of these. Some eukaryotes, in particular the fungi, arealso resistant to environmental extremes. For example, various speciesof psychrotropic fungi (i.e., Humicola marvinii; H. fuscoatra; seeWeinstein et al., Mycologia, 1997, 89(5), p. 706-711) live in thefellfield soils of the maritime Antarctic, and the larva of the brinefly (Ephydra) is adapted for life in the pink salt lakes of California'sSierra Nevada mountains.

Robust cells may be employed as single-cell biosensors, or as part of amulti-celled organism that is taken into the field. For example, yeastcells comprising the appropriate olfactory detection and signalingcomponents may be brought into the area to be tested for olfactants, orliving organisms which have had one or all of their cells converted intobiosensors may be used.

Robust cells useful in the biosensors and methods of the presentinvention therefore include prokaryotic cells such as bacterial cells,and eukaryotic cells including yeast cells, fungal cells, insect cells,nematode cells, and plant or animal cells. Biosensors constructed fromeukaryotic cells may be single-cell or may comprise intact multicellularorganisms, including nematodes (e.g., Caenorhabditis elegans),transparent or semi-transparent animals (e.g., zebrafish), and insects(e.g., Drosophila).

Yeast are perhaps the most well-known of the eukaryotes that are highlyresistant to adverse environmental conditions. Yeast cells are resistantto radiation, wind shear, and dehydration damages. Their nutrientrequirements are quite simple and minimal. In addition, yeast cells aregenetically well characterized and thus are amenable to geneticmanipulations. Furthermore, yeast cells have signaling pathwaysanalogous to those of higher animals, and hence the cells can begenetically altered to express functional signaling units of higheranimals. Yeast cells, in particular from strains of Saccharomycescerevisiae, are preferred for constructing the present biosensors. Forease of discussion, the invention will be illustrated with reference toyeast; however, it is understood that other robust cells may be used.

The yeast cells may be transfected to contain one or more exogenoussignaling pathways for transducing the signal produced by an olfactoryreceptor protein upon binding of an olfactant. Any G-protein signalingpathway may be used. In a preferred embodiment, the pathway comprisesthe G-protein-mediated activation of adenylate cyclase with resultantproduction of cAMP as a second messenger (see The Encyclopedia ofMolecular Biology, [Kendrew J and Lawrence E, eds.], Blackwell Sciences,1994, London, pp. 998-1003, herein incorporated by reference).

As shown in FIG. 1, a typical signaling unit comprises the olfactoryreceptor coupled to a signaling pathway comprising a G protein (e.g., anolfactory receptor G-protein, or Golf), adenylyl cyclase, the secondmessenger cyclic AMP (cAMP), and cAMP-activated cation channel. TheG-protein consists of three subunits; the G_(alpha) subunit (whichdictates the coupling specificity) and G_(beta) and G_(gamma) subunits.A G-protein may comprise subunits from the same source; for example,alpha, beta and gamma subunits from a single species. Alternatively, aG-protein may comprise subunits from different sources; for example,alpha, beta and gamma subunits each from a different species, or analpha subunit from one species and a beta and gamma subunit from asecond species.

In a particularly preferred embodiment, the exogenous signaling pathwaycomprises the cAMP signal transduction pathway associated with aG-protein comprising the rat M4 olfactory G-protein (Golf) alphasubunit. See Jones D T and Reed R R (1989), Science 244, 790-795 andGenBank record accession No: M26718, the disclosures of which are hereinincorporated by reference in their entirety. The rat M4 Golf alphasubunit may be associated with rat beta and gamma subunits to form theG-protein, or may be associated with beta and gamma subunits from otherspecies (e.g., Homo sapiens) to form the G-protein.

The nucleotide and amino acid sequences for H. sapiens G-protein betasubunit are found in GenBank record accession no. X04526; see alsoCodina J et al. (1986) FEBS Lett. 207 (2), 187-192, the disclosures ofwhich are herein incorporated by reference in their entirety. Thenucleotide and amino acid sequences for H. sapiens G-protein gammasubunit are found in GenBank record accession no. AF188178; see alsoHurowitz E H et al. (2000), DNA Res. 7 (2), 111-120, the disclosures ofwhich are herein incorporated by reference in their entirety.

Binding of olfactant to the expressed olfactory receptor on the surfaceof the host cell sends a signal which activates the G-protein (forexample, one comprising rat M4 Golf alpha subunit), which in turnstimulates adenylyl cyclase. The activated adenylyl cyclase catalyzesthe formation of cyclic adenosine monophosphate (cAMP) which then opensa cAMP-gated cation (usually Ca⁺⁺) channel. The sequential activation ofthe G-protein and adenylate cyclase upon binding of an olfactant to theolfactory receptor is an example of signal transduction. That is, thesignal produced by binding of olfactant to the receptor is transducedinto a biological effect by activation of the G-protein and adenylatecyclase, with subsequent production of second messenger.

In situ, the resultant influx of Ca⁺⁺ can cause the depolarization ofthe olfactory neuron; in the present biosensors, the Ca⁺⁺ influx mayactivate a signal reporter. For ease of discussion, the invention willbe illustrated with the G-protein/cAMP pathway. However, it isunderstood that any G-protein second messenger pathway may betransfected into the host cells.

A host master cell line may be constructed by the transfection,preferably sequential transfection, of genetic material encoding thenon-variable components of the signaling pathway into the host cells.The invariant components include all factors necessary for coupling ofthe signaling pathway to the chimeric olfactory receptor, and forgeneration of the signal which results in cation (e.g., Ca⁺⁺) entry intothe cell. For example, these components may comprise the G-proteinsubunits (α-, β-, and γ-subunits), type III adenylyl cyclase, and anendogenous or exogenous cation channel.

Preferably, the genetic material encoding the signaling pathwaycomponents comprises a plasmid vector. However, the genetic material maytake any form suitable for introduction into a prokaryotic or eukaryoticcell which is known in the art, for example an artificial chromosome,viral vector, liposome encapsulated DNA, etc. Preferably, the geneticmaterial stably integrates into the genome of the host cell, so that theexogenous signaling pathway components are present in subsequentgenerations.

Any suitable method may be used to transfect the host cells with thesignaling pathway components. Transfection methods for prokaryotic cellsare well known in the art, and are outlined, for example, in MolecularCloning, a Laboratory Manual, supra. Transfection methods for eukaryoticcells are also well known in the art, and include, for example, directinjection into the nucleus or pronucleus, electroporation, liposometransfer and calcium phosphate precipitation. In a preferred method, thetransfection is performed with a liposomal transfer compound, e.g.,DOTAP (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammoniummethylsulfate, Boehringer-Mannheim) or an equivalent, such asLIPOFECTIN.

Techniques for isolating the signaling pathway components, insertion ofthese components into vectors for transfecting the host cells, and forstably transfecting the host cells are within the known to those ofskill in the art. See generally Molecular Cloning, A Laboratory Manual(second edition) Ed. J. Sambrook, E. F. Fritsch, T. Maniatis (ColdSpring harbor Press. 1989). See also Jones D T and Reed R R (1989)supra; Codina J et al. (1986) supra; and Hurowitz E H et al. (2000),supra. Construction of exemplary signaling component vectors and thetransfection of S. cerevisiae to produce the yeast host master cellWIF-1 are described in Example 1.

Following the transfection of each of the signaling pathway componentsinto the host cell, expression of each component may be verified byreverse-transcription PCR (RT-PCR) as known to those skilled in the art,and as described in Example 1. The functionality of the transfectedcomponents may be tested using specific assays, for example assaysdesigned to monitor the accumulation of cAMP in response to theactivation of the olfactory signaling unit independent of receptor, asdescribed in Example 5.

A host master cell line is converted into a “pre-biosensor” bytransfection with a signal reporter, for example one responsive tointracellular levels of cAMP or Ca++. The signal reporter couples thedetection of an olfactant by the biosensor to a detectable phenomenonthat may be perceived by an observer. Preferred are signal reporterscomprising CREB and CRE-driven GFP.

The detectable phenomenon from the signal reporter may comprisefluorescent signals, although those skilled in the art will appreciatethat other indicia are known and may be used in the practice of thepresent invention, such as may be provided by labels that producesignals comprising, for example, visible light, fluorescence;radioactivity; colorimetry; X-ray diffraction or absorption; electricityor change in electric potential; or magnetism. Such labels include, forexample, fluorophores, chromophores, radioactive isotopes (e.g., ³²P or¹²⁵I) and electron-dense reagents.

Fusion proteins comprising beta-galactosidase, firefly luciferase, andbacterial luciferase segments are also known methods of detecting geneexpression and protein interactions in cells and are useful in thepresent invention. However, these methods require exogenously-addedsubstrates or cofactors, and may not be convenient in a biosensordesigned for use in the field. In the biosensors and methods of thepresent invention, therefore, an inherently fluorescent marker moleculeis preferred, since detection of such a marker requires only theradiation by the appropriate wavelength of light and is not substratelimited. A preferred inherently fluorescent molecule is GreenFluorescent Protein, or GFP.

Green Fluorescent Protein (GFP) was first isolated from the jelly fishAequorea victoria, and has an inherent green bioluminescence that can beexcited optically by blue light or nonradiative energy transfer.Sequences of GFP-encoding cDNA, and GFP proteins are known; see, e.g.,Prasher et al., Gene, 111:229 (1992), the disclosure of which is hereinincorporated by reference. Purified native GFP absorbs blue light(maximally at 395 nm with a minor peak at 470 m) and emits green light(peak emission at 509 nm) (Morise et al, Biochemistry, 13:2656 (1974);Ward et al., Photochem. Photobiol., 31:611 (1980)). It has been shownthat GFP expressed in prokaryotic and eukaryotic cells produces a stronggreen fluorescence when excited by near UV or blue light (see U.S. Pat.No. 5,491,084 to Chalfie and Prasher, herein incorporated by referencein their entirety). As this fluorescence requires no additional geneproducts from A. victoria, chromophore formation is notspecies-specific, and occurs either through the uses of ubiquitouscellular components or by autocatalysis. Expression of GFP inEscherichia coli results in an easily detected green fluorescence thatis not seen in control bacteria. See Chalfie et al., Science 263:802(1994); U.S. Pat. No. 5,491,084, supra.

As used herein, Green Fluorescent Protein or GFP refers to the variousnaturally occurring forms of GFP which can be isolated from naturalsources, as well as artificially modified GFPs which retain thefluorescent abilities of native GFP. As discussed in Ormo et al.,Science 273:1392 (1996), the disclosure of which is herein incorporatedby reference, various mutants of GFP have been created with alteredexcitation and emission maxima. Additional alterations in the GFPprotein sequence which provide inherently fluorescent, biologicallycompatible molecules will be apparent to those in the art; sequencealterations may be made to alter the solubility characteristics of theprotein, its excitation wavelength, or other characteristics, whileretaining useful fluorescent properties. See, e.g., U.S. Pat. No.5,625,048 to Tsien and Heim; WO 9711091 (Bjorn, Poulsen, Thastrup andTullin); WO 9627675 (Haseloff, Hodge, Prasher and Siemering); WO 9627027(Ward); WO 9623898 (Bjorn et al.); WO 9623810 (Heim and Tsien); WO9521191 (Chalfie and Ward), the disclosures of which are hereinincorporated by reference in their entirety.

Preferably, the genetic material comprising the signal reportercomprises one or more plasmid vectors. However, the genetic material maytake any form suitable for introduction into a prokaryotic or eukaryoticcell which is known in the art, for example an artificial chromosome,viral vector, liposome encapsulated DNA, etc. Preferably, the geneticmaterial stably integrates into the genome of the host master cell, sothat the signal reporter is present in subsequent generations.

Techniques for isolating or constructing the signal reporter, insertioninto vectors for transfecting the host cells, and for transfecting thehost cells are within the skill in the art, as discussed above.Construction of an exemplary signal reporter vector and the transfectionyeast master strain WIF-1 to produce pre-biosensor strain WIF-1α aredescribed in Example 2.

Following the transfection of the signal reporter into the host mastercell line, expression may be verified by reverse-transcription PCR(RT-PCR) as known to those skilled in the art. The functionality of thereporter may be tested, for example, by assaying for the specificresponse expected from the signal pathway upon activation of theolfactory receptor signaling pathway, as described in Example 5.

Exemplary signal reporters include, for example:

-   1. Cyclic AMP-responsive GFP expression systems—In such systems, the    cAMP generated in yeast cells upon detection of a specific molecule    through the olfactory receptor activates “cAMP-responsive element    binding protein” (CREB). The activated CREB in turn binds to a    cAMP-responsive element (CRE) sequence of DNA that is operatively    linked to nucleotide sequences encoding GFP (i. e., can drive the    expression of GFP).-   2. Cyclic AMP-responsive β-galactosidase expression systems—In such    systems, the cAMP generated in yeast cells upon activation of the    olfactory receptor activates CREB. The activated CREB in turn binds    to a CRE sequence of DNA that can drive the expression of the    bacterial lacZ gene (which encodes the β-galactosidase). When grown    in the presence of X-Gal, the cells expressing β-galactosidase turn    blue in color. Since β-galactosidase is expressed only in response    to cAMP coupled to the olfactory receptor, the blue color identifies    the detection of a specific molecule by the respective yeast colony.    This system is useful if the immediate detection of olfaction is not    desired, or is not possible, as the calorimetric response may take    several hours to develop.-   3. Ca⁺⁺-responsive luminescence reporter systems—For example, cDNAs    encoding rat olfactory cell specific Ca⁺⁺ channel and photoprotein    Apoaequorin of coelenterate jelly fish Aequorea victoria may be    expressed in S. cerevisiae. The yeast may be grown in medium    containing coelenterazine so that the Ca⁺⁺-sensitive aequorin is    formed. Upon the influx of Ca⁺⁺ after activation of the olfactory    receptor, aequorin binds to Ca⁺⁺ and emits blue light. This blue    light can be read at 460-470 nm, for example in a luminescence    bioassay plate reader.-   4. Fluorescent cytosolic Ca⁺⁺ indicators—Ca⁺⁺ influx following the    detection of a specific molecule through the olfactory receptor can    be also monitored using any fluorescent cytosolic Ca⁺⁺ indicator,    for example FURA-2-mediated Ca⁺⁺ fluorescence (510 nm).-   5. Electrophysiological systems—Such systems comprise the    electrophysiological detection of Ca⁺⁺ influx, for example by    measuring the depolarization of the biosensor's cell membrane (or    the membrane of an associated cell, such as a neuron). Such methods    are known in the art. See, for example, U.S. Pat. No. 5,993,778    which describes the measurement of the change in cell-membrane    potential of cells expressing olfactory receptors in situ with an    (termed an “electro-olfactogram recording”). This method is easily    adaptable to measure membrane potentials of biosensors located, for    example, on an assay plate or array.

A pre-biosensor may be transfected with one or more expression vectorscomprising an actual or putative hypervariable segment of an olfactoryreceptor to produce a biosensor. Transfection may be accomplished by anymethod designed to introduce genetic material into a prokaryotic oreukaryotic cell, as described above.

Multicellular organisms comprising biosensors may be produced bytransfecting the signaling pathway components, signal reporter and anexpression vector comprising an olfactory receptor hypervariable segment(see below) into a pluripotent stem cell, for example an embryonic stemcell, and allowing the transfected stem cell to develop into a matureorganism. Such techniques are known in the art; see for example Thomsonet al., (1998) Science 282:1145-47 and Reubinoff et al. (2000) Nat.Biotechnol. 18: 399-404 (both incorporated herein by reference). Forexample, chimeric animals may be produced by aggregation of altered stemcells with normal blastocyst cells and transgenic animals are recoveredas offspring of the chimeric animals, according to the method ofCapecchi, M. R., 1989, Science 244: 1288, the entire disclosure of whichis incorporated herein by reference.

Alternatively, transfection of lower eukaryotes (e.g., Caenorhabditiselegans) to create multicellular organisms comprising biosensors may beperformed by direct injection of genetic material into the germ cells orgonads, according to known techniques.

An expression vector transfected into the pre-biosensor may comprise anexpression cassette comprising sequences encoding a chimeric olfactoryreceptor. The encoded chimeric olfactory receptor may comprise aprocessing/transport and G-protein receptor coupling segment operativein an olfactory receptor, and an olfactory receptor hypervariablesegment. In preferred embodiments, the processing/transport andG-protein receptor coupling segments are N- and C-terminal to anolfactory receptor hypervariable segment, respectively. Theprocessing/transport segment N-terminal to the olfactory receptorhypervariable region directs its processing and transport of thechimeric olfactory receptor, and the coupling segment C-terminal to theolfactory receptor hypervariable region couples the chimeric olfactoryreceptor to an exogenous signaling pathway in the pre-biosensor cellline.

The basic expression vector (i.e., the vector comprising a cloningcassette and nucleotide sequences encoding the processing/transport andcoupling segments before addition of nucleotide sequences encoding anolfactory receptor hypervariable segment) is considered part of thepresent invention. Construction of an exemplary basic expression vectorfor yeast, comprising a cloning cassette and nucleotide sequencesencoding processing/transport and coupling segments derived from the ratRI7 olfactory receptor is given in Example 4.

As the olfactory receptor hypervariable segment (i.e., transmembranedomains II-VII) is involved in ligand detection and discrimination, alibrary of expression vectors containing different olfactory receptorhypervariable segments may be constructed which encode chimericolfactory receptors of different specificities. This expression librarymay then be used to construct a library of biosensors of differingspecificity, which are then screened for the ability of individualbiosensors of the library to detect an olfactant of interest.

The segment N-terminal to the hypervariable region of the chimericolfactory receptor preferably comprises amino acid sequences derivedfrom an olfactory receptor of the host cell species. For example, if thehost master cell line is derived from S. cerevisiae, processing andtransport segments from segments N-terminal to the hypervariable segmentof a naturally occurring S. cerevisiae olfactory receptor may be used.An example of such a segment is the first 60 amino acids of the S. pombemam2 pheremone receptor, represented below with the N-terminus to theleft, and the C-terminus to the right:

(SEQ ID NO: 1) MRQPWWKDFTIPDASAIIHQNITIVSIVGEIEVPVSTIDAYERDRLLTGMTLSAQLALGV

The full length cDNA and protein translation of mam2 is given in SEQ IDNO: 28 and SEQ ID NO: 29, respectively, and in GenBank record accessionno. X61672; see also Kitamura K and Shimoda C (1991), EMBO J. 10 (12),3743-375, the disclosures of which are herein incorporated by referencein their entirety.

Processing/transport segments from another species' olfactory receptormay also be used if it is known or determined that the segments areoperational in the host master cell line. For example, the inventorshave discovered that the N-terminal 61 amino acids of the rat RI7olfactory receptor, represented below with the N-terminus to the left,and the C-terminus to the right, allows the efficient processing andtransport of a chimeric olfactory receptor in yeast host cells:

MERRNHSGRVSEFVLLGFPAPAPLRVLLFFLSLLXYVLVLTENMLIIIAIR (SEQ ID NO: 2)NHPTLHKPMY

The full length cDNA and protein translation for the rat RI7 olfactoryreceptor are given in SEQ ID NO: 30 and SEQ ID NO: 31, respectively. Seealso GenBank record accession no. M64386 and Buck and Axel (1991), Cell65: 175-187, the disclosures of which are herein incorporated byreference in their entirety.

Thus, suitable processing/transport segments may be identified bothstructurally (i.e., they are located N-terminal to the hypervariablesegment of olfactory receptor proteins) and functionally (i.e., theydirect efficient processing and transport of the chimeric olfactoryreceptors to the host cell membrane).

The coupling segment C-terminal to the hypervariable segment of thechimeric olfactory receptor preferably comprises sequences derived fromsegments C-terminal to the hypervariable segment of an olfactoryreceptor known to couple efficiently with an exogenous signaling pathwaytransfected into the pre-biosensor cell line. Preferably, the couplingsegment will comprise amino acid segments of olfactory receptors of thesame species from which the exogenous signaling pathway was obtained.For example, for a yeast cell engineered with a rat signaling pathwaycomprising rat M4 Golf-mediated activation of adenylyl cyclase, thecoupling segment of the chimeric receptor protein may comprise the last35 amino acids of the rat RI7 olfactory receptor, represented below withthe N-terminus to the left, and the C-terminus to the right:

IIYCLRNQDVKRALRRTLHLAQDQEANTNKGSKIG (SEQ ID NO: 3)

Other coupling segments may also be used, if it is known or determinedthat they promote efficient coupling to the exogenous signaling pathwayin the pre-biosensor.

Suitable coupling segments may therefore be identified both structurally(i.e., they are located C-terminal to the hypervariable segment ofolfactory receptor proteins) and functionally (i.e., they couple thechimeric olfactory receptor to a specific G-protein second messengerpathway).

Variants of a given processing/transport or coupling segment may existin nature or may be artificially produced. These variants may be allelicvariations characterized by differences in the nucleotide sequences ofthe structural gene coding for the protein, or may involve differentialsplicing or post-translational modification. The skilled artisan canisolate or produce derivatives of an processing/transport or couplingsegments having single or multiple amino acid substitutions, deletions,additions, or replacements. These derivatives may include, inter alia:(a) derivatives in which one or more amino acid residues are substitutedwith conservative or non-conservative amino acids, (b) derivatives inwhich one or more amino acids are added to the protein, (c) derivativesin which one or more of the amino acids includes a substituent group,and (d) derivatives in which the protein is fused with another peptide.The techniques for obtaining these derivatives, including genetic(suppressions, deletions, mutations, etc.), chemical, and enzymatictechniques, are known to persons having ordinary skill in the art.

The invention thus provides a chimeric olfactory receptor proteincomprising an actual or putative olfactory receptor hypervariablesegment and defined segments N- and C-terminal to the hypervariablesegment, wherein the segment N-terminal to the hypervariable segmentdirects the efficient processing and transport of the chimeric receptorin the host cell, and the segment C-terminal to the hypervariablesegment couples the chimeric receptor to an exogenous signaling pathwayof the host cell.

In a preferred embodiment, the chimeric olfactory receptor comprises anolfactory receptor hypervariable segment, a yeast mam2processing/transport segment N-terminal to the hypervariable segment,and a rat R17 receptor coupling segment C-terminal to the hypervariableregion. The yeast mam2 segment may include the first 60 amino acids ofthe mam2 protein. The yeast mam2 segment ensures maximal expression andproper translocation of the chimeric olfactory receptor protein in yeasthost cells, and the rat RI7 segment restricts the cell signalinginteraction of the chimera to the exogenous Golf-mediated cAMP pathwayof the yeast master cell. In a particularly preferred embodiment, theyeast mam2 segment is replaced by the N-terminal 61 amino acidscomprising the processing/transport segment of the rat RI7 receptor (seeExample 4).

Actual or putative hypervariable segments for cloning into the basicexpression vector may be obtained by any method designed to isolate thenucleic acid sequences encoding transmembrane domains II through VII;for example, nuclease digestion of appropriate olfactory receptorgenomic or cDNA sequences or PCR amplification of the hypervariablesegment.

A preferred method is PCR amplification of transmembrane domains II-VIIbased on the flanking consensus amino acid sequences known to exist inolfactory receptors. See Buck and Axel (1991), Cell 65: 175-187;Krautwurst et al. (1998), Cell 95: 917-926 and Zhao et al. (1998),Science 279: 237-242, the disclosures of which are herein incorporatedby reference.

Appropriate degenerate primers may be synthesized based on the consensussequence of the species of interest. For example, the PCR reaction maybe carried out on a cDNA library made from yeast, C. elegans,Drosophila, murine, canine, human or other olfactory or sensory cells.The PCR-products may be directly ligated into the basic expressionvector cloning cassette, thereby generating vectors encoding chimericreceptors as shown in FIG. 3. Exemplary degenerate primers for PCRamplification of olfactory receptor hypervariable segments from the ratare given below in Table 1. Exemplary degenerate primers for PCRamplification of olfactory receptor hypervariable segments from themouse are given below in Table 2.

TABLE 1 Exemplary degenerate primers for amplification of olfactoryreceptor hypervariable segments from the rat* In the following primers,“I” is inosine. Forward Primers5′-AA(T/C)T(G/A)(G/C)ATI(C/A)TI(G/C)TIAA(T/C)(C/T)TIGCIGTIGCIGA-3′ (SEQID NO: 4)5′-AA(T/C)TA(T/C)TT(T/C)(C/A)TI(G/A)TIAA(T/C)CTIGCI(T/C)TIGCIGA-3′ (SEQID NO: 5)5′-AA(T/C)(T/C)(T/A)ITT(T/C)(A/C)TIATI(T/A)CICTIGCIT(G/C)IGCIGA-3′ (SEQID NO: 6)5′-(C/A)GITTI(C/T)TIATGTG(T/C)AA(C/T)CTI(T/A)(G/C)(C/T)TT(T/C)GCIGA-3′(SEQ ID NO: 7)5′-ACIGTITA(T/C)ATIACICA(T/C)(C/T)TI(A/T)(C/G)IATIGCIGA-3′ (SEQ ID NO:8) Reverse Primers5′CTGI(C/T)(G/T)(G/A)TTCATIA(A/T)I(A/C)(C/A)(A/G)TAIA(T/C)IA(T/C)IGG(G/A)TT-3′(SEQ ID NO: 9)5′-(G/T)(A/G)T(C/G)(G/A)TTIAG(A/G)CA(A/G)CA(A/G)TAIATIATIGG(G/A)TT-3′(SEQ ID NO: 10) 5′-TCIAT(G/A)TT(A/G)AAIGTIGT(A/G)TAIATIATIGG(G/A)TT-3′(SEQ ID NO: 11)5′-GC(C/T)TTIGT(A/G)AAIATIGC(A/G)TAIAG(G/A)AAIGG(G/A)TT-3′ (SEQ ID NO:12)5′-AA(A/G)TCIGG(G/A)(C/G)(T/A)ICGI(C/G)A(A/G)TAIAT(C/G)AIIGG(G/A)TT-3′(SEQ ID NO: 13)5′-(G/C)(A/T)I(G/C)(A/T)ICCIAC(A/G)AA(A/G)AA(A/G)TAIAT(A/G)AAIGG(G/A)TT-3′(SEQ ID NO: 14) *Adapted from Buck and Axel, supra.

Each of the five forward primers and each of the six reverse primersfrom Table 1 may be used simultaneously in PCR amplification reactionsof reverse-transcribed rat olfactory epithelial RNA to obtain ratolfactory receptor hypervariable segment cDNAs. Appropriate PCRamplification conditions are known to those skilled in the art, forexample as outlined in Buck and Axel, supra.

TABLE 2 Exemplary degenerate primers for amplification of olfactoryreceptor hypervariable segments from the mouse** In the followingprimers, “P” is dP-CE phosphor- amidite (6H,8H-3,4-dihydro pyrimido[4,5-c][1,2] oxazin-7-one,8-[(5′-dimethoxytrityl-β-Ddeoxyribo-furanosyl), 3′-[(2-cyanoethyl)-(N,N- diisopropyl)]-phosphoramidite).This compound is available from Glen Research (Sterling, VA). ForwardPrimer 5′-GGGGTCCGGAG(A/G)(C/G)T(A/G)A(A/G/T)AT(A/G/P)A(A/G/P)(A/G/P)GG-3′ (SEQ ID NO: 15) Reverse Primer5′-GGGGCTGCAGACACC(A/C/G/T)ATGTA(C/T)(C/T)T(A/C/ G/T)TT(C/T)(C/T)T-3′(SEQ ID NO: 16) **Adapted from Krautwurst et al., supra.

The primers in Table 2 may be used in PCR amplification reactions ofreverse-transcribed mouse olfactory epithelial RNA to obtain mouseolfactory receptor hypervariable segment cDNAs. Appropriate PCRamplification conditions are known to those skilled in the art, forexample as outlined in Krautwurst et al., supra.

Different variants of a given olfactory receptor protein may exist innature. These variants may be allelic variations characterized bydifferences in the nucleotide sequences of the structural gene codingfor the protein, or may involve differential splicing orpost-translational modification. The skilled artisan can isolate orproduce derivatives of an olfactory receptor protein hypervariablesegment having single or multiple amino acid substitutions, deletions,additions, or replacements. These derivatives may include, inter alia:(a) derivatives in which one or more amino acid residues are substitutedwith conservative or non-conservative amino acids, (b) derivatives inwhich one or more amino acids are added to the protein, (c) derivativesin which one or more of the amino acids includes a substituent group,and (d) derivatives in which the protein is fused with another peptide.The techniques for obtaining these derivatives, including genetic(suppressions, deletions, mutations, etc.), chemical, and enzymatictechniques, are known to persons having ordinary skill in the art. See,for example, WO 97/35985, the disclosure of which is herein incorporatedin its entirety, which describes methods of producing mutant olfactoryreceptor hypervariable segments.

Libraries of the chimeric olfactory receptor cDNAs may be generated bycloning the cDNAs into a basic expression vector, which may bepropagated in an appropriate E. coli strain. Such libraries may be usedto produce libraries of biosensors, by transfecting a yeastpre-biosensor strain containing an appropriate exogenous signalingpathway (e.g., WIF-1α, as described in Example 2).

The biosensors of the invention, either individually or in a library,may be tested to determine their ability to detect a given olfactant.The appropriate test for determining olfactant specificity depends onthe signal reporter of the biosensor. For example, if the biosensorsignal reporter comprises β-galactosidase, the biosensors may be grownin the presence of the olfactant and colony color developed by soft agaroverlay using the chromogenic substrate X-gal. If the biosensor signalreporter comprises GFP, the biosensors may be grown in 96-well platesand the expression of GFP in response to the chemical agents of interestcan be monitored.

Green fluorescent protein expressing yeast colonies or blue yeastcolonies (i.e., those expressing olfactory receptor-drivenβ-galactosidase) representing biosensors of interest may be rescued fromthe primary plating, and subjected to multiple rounds of secondaryvalidation assays to confirm sensitivity and specificity of activationfor each biosensor. Confirmed positive clones may be subjected to DNAsequence analysis. Selected biosensors with defined olfactory ligandspecificity may then be assembled into specific arrays for olfactantdetection.

The methods for detecting biosensors of the present invention that candetect a given olfactant may be automated to provide convenient, realtime, high volume methods of identifying biosensors with specificaffinity for an olfactant. A test sample or environment may also betested for the presence of an olfactant using one or more biosensors ofknown specificity. Preferably, automated methods are designed to detectthe change in intracellular cAMP or Ca⁺⁺ in the biosensor, as will beapparent to those skilled in the art.

The invention thus provides a method of identifying biosensors which candetect a given olfactant, comprising the steps of providing at least onebiosensor of the invention, wherein detection of the olfactant by thebiosensor generates a detectable phenomenon from the signal reportercontacting the one or more biosensor with the olfactant; and observingwhether the detectable phenomenon is produced from the signal reporter.

The invention further provides a method for detecting a selectedolfactant in a sample, comprising the steps of providing one or morebiosensors of the invention capable of detecting the olfactant, whereindetection of the olfactant by the biosensor generates a detectablephenomenon from the signal reporter; contacting the one or morebiosensors with a sample suspected of containing the olfactant; andobserving whether the detectable phenomenon is produced from thebiosensor signal reporter. The sample to be tested may be liquid, gas,or a mixture of liquid and gas (e.g., air mixed with water vapor), or besusceptible to conversion into a liquid or gas (e.g., by volatilization,sublimation, melting, vaporization, or the like).

The detectable phenomenon may comprise fluorescence if the detectablemolecule is a fluorescent indicator such as GFP. Other optical indiciathat are suitable for real-time or long-term measurement of olfactionmay also be used, as will be apparent to those skilled in the art.

Preferably the detectable phenomenon from the signal reporter isdetected by an apparatus capable of detecting the phenomenon, forexample an automated fluorescence plate reader. Generally an apparatususeful in the invention will comprise a measurement tool, such as afluorescence measurement tool, for measuring the detectable phenomenon,means for controlling the measurement tool, such as a computercontroller, and one or more biosensors of the invention (preferablycomprising an array or biochip; see below). Measurement points may beover time, or among test and control biosensors.

A computer program product may control operation of the computercontroller driving the measurement tool. A preferred computer programproduct comprises a computer readable storage medium havingcomputer-readable program code means embodied in the medium. Hardwaresuitable for use in such automated apparatus will be apparent to thoseof skill in the art, and may include, automated sample handlers,printers and optical displays. The measurement tool may contain one ormore photodetectors for measuring the fluorescence signals from sampleswhere fluorescently detectable molecules are utilized. The measurementtool may also contain a computer-controlled stepper motor so that eachbiosensor can be arranged in an array and automatically and repeatedlypositioned opposite a photodetector during the step of signal detection.

The measurement tool is preferably operatively coupled to a generalpurpose or application specific computer controller. The controllerpreferably comprises a computer program product for controllingoperation of the measurement tool and performing numerical operationsrelating to the above-described steps. The controller may accept set-upand other related data via a file, disk input or data bus. A display andprinter may also be provided to visually display the operationsperformed by the controller. It will be understood by those having skillin the art that the functions performed by the controller may berealized in whole or in part as software modules running on a generalpurpose computer system. Alternatively, a dedicated stand-alone systemwith application-specific integrated circuits for performing the abovedescribed functions and operations may be provided. Preferably, thestand-alone detection system is portable, and may be operated in thefield under adverse conditions.

An array useful with the above-described apparatus may comprise a solidsupport carrying biosensors in fixed positions or cells. The appropriatepattern or distribution of biosensors in the array depends on theparticular application, and can readily be determined by one of ordinaryskill in the art.

The solid substrate onto which the biosensors are fixed or contained maycomprise, for example, organic or inorganic substrates such as glass,polystyrenes, polyimides, silicon dioxide and silicon nitride. Othersuitable substrates are known to those skilled in the art.

The arrays may be in the form of “biochips” comprising a set pattern ofbiosensors arranged on a substrate, optionally together with machinereadable information encoded on the substrate. For example, the machinereadable information may concern the location and type of biosensors onthe chip. The machine readable information may be magnetic, optical(e.g., a bar code) or laser-based. The biochips may be in any convenientshape or size which allows it to be exposed to olfactants and read by adetector.

The present biosensors may also be arranged on or contained in aportable container, such as a vial or badge, that may be carried or wornby humans working an environment contaminated by noxious or toxicchemicals. The portable container may then be placed in an detectionapparatus as described above to determine whether the badge (andtherefore the bearer) has been exposed. In another embodiment, theportable container may comprise a built-in detection system, so that thebearer is immediately informed of an exposure.

It is understood that various combinations of the components forconstructing the biosensors of the invention may be provided in kits.For example, one or more pre-biosensor cell lines and a basic expressionvector as described above (i.e., an expression vector comprising acloning cassette and sequences encoding defined olfactory receptor N-and C-terminal segments, as described above) may be provided.Optionally, the appropriate instructions and reagents for cloningolfactory receptor hypervariable segments into the basic expressionvector, and for transfecting the master cell lines with these expressionvectors, may be included in the kit.

Other kits may comprise the individual signaling pathway components forconstructing a host master cell line as described above, for example inthe form of plasmid vectors comprising nucleic acid sequences encodingthe signaling pathway components, and a basic expression vector asdescribed above. Again, the appropriate reagents and instructions foruse may optionally be included. In a variation of this kit design, thekit may instead comprise one or more host master cell lines and one ormore signal reporters for transfection into a master cell line toproduce pre-biosensor cells. With such a kit, a user would be able tochoose a signal reporter which suited his needs.

Further kits may comprise one or more fully constructed biosensors withor without defined olfactant specificities, for example in the form of alibrary to be screened. Again, the appropriate reagents and instructionsfor use may optionally be included.

The practice of invention is illustrated by the following non-limitingexamples.

EXAMPLE 1 Construction of Yeast Master Strain WIF-1

The non-variable components of the cAMP signal transduction pathwayconsisting of the rat M4 Golf alpha subunit, the human G-protein β- andγ-subunits, and type III adenylyl cyclase (AC) were sequentiallyintegrated into the yeast cell using two distinct yeast expressionvectors derived from the yeast expression vector pESC. These vectorswere:

vector component 1. pESCtrp-Golf-cyc-DH5α rat M4 Golf α-subunit;adenylate cyclase 2. pESCura-β2γ5-DH5α human G-protein β- and γ-subunits

Vectors 1 and 2 were deposited on Feb. 1, 2001 with the patent culturecollection of the National Center for Agricultural Utilization Research,Agricultural Research Service, U.S. Department of Agriculture (NRRL),1815 North University Street, Peoria, Ill. 61604 U.S.A., in accordancewith the Budapest Treaty, and have been assigned accession nos. NRRLB-30412 and NRRL B-30414, respectively. Master yeast strain WIF-1 wasdeposited on Feb. 7, 2001 with the NRRL in accordance with the BudapestTreaty; accession no. NRRL Y-30415.

1.1—Construction of the Yeast Expression Vectors

pESCtrp-Golf-cyc-DH5α—Rat M4 Golf α-subunit (Gαolf) was cloned from ratolfactory epithelial lamellae total RNA using RT-PCR. Reversetranscription of the mRNA into cDNA was carried out using theReady-To-Go™ T-Primed First-Strand Kit manufactured by AmershamPharmacia Biotech Inc USA, using the manufacturer's protocol. Thefollowing primers were used for the subsequent PCR of Gαolf:

Forward (sense): 5′-AGCCAGCAGGCATGGGGTGTTTGG-3′ (SEQ ID NO: 17) Reverse(antisense): 5′-TCACAAGAGTTCGTACTGCTTGAG-3′ (SEQ ID NO: 18)

The PCR cycling protocol was 1×2 min at 94° C.; 30×1 min each at (94°C., 60° C., 72° C.); 1×10 min. at 72° C. using pfuTURBO DNA polymerase.

The PCR product containing the Gαolf sequence and the 5′-upstream Kozaksequence was inserted into sub-cloning vector pCR2.1 (Invitrogen). Afterchecking the orientation of the inserted Gαolf cDNA by restriction andsequence analyses, the insert in the right orientation was excised frompCR2.1 vector by Not I and Sac I. The excised and gel purified Not I-SacI Gαolf insert was cloned into yeast expression vector pESC-trp(Stratagene) digested with Not I and Sac I to produce pESCtrp-Golf. Thepresence of the Gαolf sequence in pESCtrp-Golf was verified by DNAsequencing. pESCtrp-Golf was propagated in E. coli DH5α, and the plasmidwas purified for the subsequent insertion of the cDNA encoding adenylylcyclase.

The cloning vector pBSK KS containing olfactory adenylyl cyclase (AC)type III (obtained from Dr. Randall R. Reed, Johns Hopkins) waspropagated in E. coli DH5α and the plasmid was purified by standardtechniques. The cDNA sequence encoding AC was excised from pBSK KS bydigestion with Apa I and Nhe I. The Apa I-Nhe I AC fragment was clonedinto pESCtrp-Golf that had been digested with Apa I and Spe I to producepESCtrp-Golf-cyc-DH5α. pESCtrp-Golf-cyc-DH5α was propagated in E. coliDH5α.

pESCura-β2γ5-DH5α—The cDNA encoding human G-protein beta subunit β2 wasexcised from the cloning vector pSP73 (kindly provided by Dr. ShojiOsawa, University of North Carolina at Chapel Hill) using restrictionenzymes Hind III-EcoRI, and ligated into the Sal I-Xho I linearizedyeast expression vector pESC-ura The cDNA sequence for the humanG-protein beta subunit β2 is given in SEQ ID NO: 32, and the predictedamino acid sequence is given in SEQ ID NO: 33. See also GenBank recordaccession no. X04526, the disclosure of which is herein incorporated byreference. The orientation of the β2-insert was confirmed by restrictionand sequence analyses. In the resultant vector, named pESCura-β2, thecDNA for human G-protein gamma subunit γ5 was introduced as follows: ThecDNA insert encoding human γ5 was excised from pBluescript SK (kind giftfrom Dr. Janet Robishaw, Geisinger Clinic, Danville, Pa.) using therestriction enzymes Pvu I and Eco RI. The human G-protein γ5 cDNAsequence is given in SEQ ID NO: 34, and the predicted amino acidsequence is given in SEQ ID NO: 35. See also GenBank record accessionno. AF188178, the disclosure of which is herein incorporated byreference. The Pvu I-Eco RI cDNA fragment containing human γ5 was clonedinto pESCura-β2 digested with Eco RI-Pac I sites by blunt-end ligationto produce pESCura-β2γ5-DH5α, and propagated in DH5α.

1.2—Transfection of S. cerevisiae with the Yeast Expression Vectors toForm Yeast Host Master Cell WIF-1

Yeast cells competent for transfection were prepared as follows: Severalcolonies of the YPH 501 strain of the yeast S. cerevisiae, which is anauxotrophic mutant for the amino acids Trp, Leu, Ura, and His, wereinoculated into 50 ml of YPD medium (1% yeast extract, 2% Bacto peptone,2% dextrose, and 0.0075% adenine-hemisulfate in 1 liter of distilledwater), and incubated at 30° C. for 16-18 hr with shaking at 200 rpm toobtain stationary phase cultures (OD₆₀₀ greater than 1.5). Thirty ml ofthe stationary culture was transferred to 300 ml of fresh YPD medium,and was regrown at 30° C. for 3 hr with shaking at 200 rpm until theOD₆₀₀ reached 0.4-0.6. Cells competent for transfection were collectedby centrifugation (1000×g for 5 min at room temperature) and the cellpellet was washed by resuspension in 25-50 ml of sterile TE (Tris 10 mM,pH 7.5 and 1 mM EDTA) followed by centrifugation. The competent cellswere then resuspended in 1.5 ml of freshly prepared sterile TE/LiAcsolution (100 mM lithium acetate in TE).

Yeast expression vectors pESCtrp-Golf-cyc (100 ng) and pESCura-β2γ5 (100ng) and 0.1 mg of carrier DNA (herring testes DNA) in a total volume of0.015 ml were placed in a 1.5 ml tube into which 0.1 ml of competentcells were added and mixed thoroughly. In to this mixture, 0.6 ml ofsterile PEG/LiAc solution (40% PEG-4000 in TE/LiAc solution) was addedand mixed by vortexing for 10 sec. Following a brief incubation for 30min at 30° C. in a shaking incubator, 0.07 ml of DMSO was added and thetube contents were gently mixed. The tubes were then subjected toheat-shock (42° C.) for 15 min, after which each tube was chilled in anice-water bath for 2 min. The transfected cells were collected bycentrifugation (14000 g for 5 sec at room temperature) and the cellpellet was resuspended in 0.5 ml of sterile TE buffer.

An aliquot (0.1-0.2 ml) of the cells were plated onto a 100-mm petridish containing SD trp, ura amino acid drop-out medium (6.7 g yeastnitrogen base without amino acids, 20 g glucose, 20 g Agar and 1 Literwater containing L-Isoleucine, 30 mg/l; L-Valine, 150 mg/l; L-AdenineHemisulfate, 20 mg/l; L-Arginine, 20 mg/l; L-histidine, 20mg/l;L-methionine, 20 mg/l; L-tyrosine, 30 mg/l, L-threonine 200 mg/l;L-Phenylalanine, 50 mg/l, L-lysine, 30 mg/l; and L-leucine, 100 mg/l,but does not contain L-Tryptophan and L-Uracil). Thus, the SD trp, uraamino acid drop-out medium selects for transfected cells harboring bothpESCtrp-Golf-cyc and pESCura-β2γ5 expression vectors.

The petri-dishes were incubated at 30° C. for 2-4 days or until coloniesappeared. The larger colonies were picked and grown on fresh selectiveSD trp, ura amino acid drop-out medium (agar dishes as well as liquidmedium). A yeast host master cell containing pESCtrp-Golf-cyc andpESCura-β2γ5 expression vectors was isolated and named WIF-1. This hostmaster cell was stored as glycerol stocks (25%) at −80° C.

EXAMPLE 2 Construction of Yeast Reporter Strain WIF-1α

The WIF-1 host master cell described above can be transfected withsignal reporters of different types to form pre-biosensor cells.Transfection vectors comprising three separate signal reporters (for theconstruction of three separate pre-biosensor cells) have beenconstructed. These vectors were:

vector component 3. pESChis-creb-cregfp-DH5α CREB; CRE-driven GFP 4.pESChis-creb-cregal-DH5α CREB; CRE-driven beta galactosidase 5.pESChis-ONGC-aeq-DH5α Ca⁺⁺ channel; Apoaequorin

Vector 3 (pESChis-creb-cregfp-DH5α) was deposited on Feb. 1, 2001 withthe patent culture collection of the National Center for AgriculturalUtilization Research, Agricultural Research Service, U.S. Department ofAgriculture (NRRL), 1815 North University Street, Peoria, Ill. 61604U.S.A., in accordance with the Budapest Treaty, and has been assignedaccession no. NRRL B-30413.

2.1—Construction of the Yeast Expression Reporter VectorpESChis-creb-cregfp-DH5α

pESChis-creb-cregfp-DH5α: This construct was made using the yeastexpression vector, pESC-HIS. The CRE-driven GFP was inserted in place ofthe pESC-HIS GAL1 promoter; to this end, the GAL1 promoter of pESC-HISwas excised by digestion with Bam HI and Age I. pESC-His was religatedto form the circularized pESCDG1-HIS vector.

The cDNA encoding human CREB was excised from pCMV-CREB vector(Clontech, Palo Alto, Calif.) by digestion with Eco RI and Xba I, andligated into pESCDG1-HIS linearized by digestion with Eco R I-Spe I. Theresultant vector was named pESCDG1-His-CREB.

A modified cDNA encoding GFP under the control of human CRE promoter wasobtained from pCRE-d2EGFP vector by PCR amplification. The PCR primerswere designed so that the resulting PCR product lacked the destabilizingPEST-sequence (introduced into pCRE-d2EGFP by Clontech to monitordynamic changes in GFP-expression because it enhances degradation of theprotein) and contained a new stop-codon. The PCR primers were:

Forward (sense) primer: 5′-TAGGTACCGAGCTCTTACGCGTGCTAGCGCA-3′ (SEQ IDNO: 19) Reverse (antisense) primer:5′-GCTCTAGATTACTTGTACAGCTCGTCCATGCCGAG-3′ (SEQ ID NO: 20)

The PCR cycling protocol was 1×2 min. at 94° C.; 30×1 min. each at (94°C., 60° C., 72° C.); 1×10 min. at 72° C. using pfuTURBO DNA polymerase.The PCR product containing the CRE and GFP cDNA in tandem was clonedinto pESCDG1-His-CREB vector by blunt-end ligation into the Sma I site,after the cut-ends of the vector had been filled in. The orientation andthe sequence of the CRE-GFP insert were verified by DNA sequencing. Thevector was named pESChis-creb-cregfp, and propagated in DH5α.

2.2—Transfection of WIF-1 with pESChis-creb-cregfp-DH5α to FormPre-biosensor WIF-1α

Several colonies of WIF-1 host master cell (described above) were grown50 ml of YPD medium (see above) and incubated at 30° C. for 16-18 hrswith shaking at 200 rpm to obtain stationary phase cultures (OD₆₀₀greater that 1.5). Thirty ml of the stationary cultures were transferredto 300 ml of YPD medium and regrown at 30° C. for 3 hr with shaking (200rpm) until the OD₆₀₀ reached 0.4-0.6. The host master cells werecollected by centrifugation (1000×g for 5 min at room temperature) andthe cell pellet was washed by resuspension in 25-50 ml of sterile TEfollowed by centrifugation. The host master cells were resuspended in1.5 ml of freshly prepared sterile TE/LiAc solution (see above) inpreparation for transfection.

Yeast expression vector pESChis-creb-cregfp (100 ng) and 0.1 mg ofcarrier DNA (herring testes DNA) in a total volume of 0.015 ml wasplaced in a 1.5 ml tube into which 0.1 ml of WIF-1 host master cells hadbeen added, and mixed thoroughly. Into this mixture, 0.6 ml of sterilePEG/LiAc solution (see above) was added and mixed by vortexing for 10sec. Following a brief incubation for 30 min at 30° C. in a shakingincubator, 0.07 ml of DMSO was added and the tube contents gently mixed.The tubes were then subjected to heat-shock (42° C.) for 15 min, afterwhich the tube was chilled in an ice-water bath for 2 min.

The transfected WIF-1 cells were collected by centrifugation (14000 gfor 5 sec at room temperature) and the cell pellet was resuspended in0.5 ml of sterile TE buffer. An aliquot (0.1-0.2 ml) of the cells wereplated onto a 100-mm petri-dish containing SD his, trp, ura drop-outamino acid medium (6.7 g yeast nitrogen base without amino acids, 20 gglucose, 20 g Agar and 1 Liter water containing L-Isoleucine, 30 mg/l;L-Valine, 150 mg/l; L-Adenine Hemisulfate, 20 mg/l; L-Arginine, 20 mg/l;L-methionine, 20 mg/l ; L-tyrosine, 30 mg/l, L-threonine 200 mg/l;L-Phenylalanine, 50 mg/l, and L-lysine, 30 mg/l, but not L-leucine,L-Histidine, L-Tryptophan and L-Uracil). Thus, the SD his, trp, uradrop-out amino acid medium selects for transfected WIF-1 cells (i.e.,pre-biosensor cells) that harbor pESChis-creb-cregfp-DH5α in addition topESCtrp-Golf-cyc-DH5α and pESCura-β2γ5-DH5α expression vectors.

The petri-dishes were incubated at 30° C. for 2-4 days or until coloniesof pre-biosensor cells appeared. The larger colonies were picked andgrown on fresh selective SD his, trp, ura drop-out amino acid medium (onagar dishes as well as in medium liquid medium). A pre-biosensortransfected with pESChis-creb-cregfp-DH5α in addition topESCtrp-Golf-cyc-DH5α and pESCura-β2γ5-DH5α expression vectors wasisolated and named WIF-1α. WIF-1α was stored as glycerol stocks (25%) at−80° C.

2.3—Transfection of WIF-1 with pESChis-creb-cregal-DH5α andpESChis-ONGC-aeq-DH5α

Yeast host master cell WIF-1 is transfected with eitherpESChis-creb-cregal-DH5α or pESChis-ONGC-aeq-DH5α as described above forpESChis-creb-cregfp-DH5α to produce prebiosensors WIF-1-ga1 andWIF-1aeq.

EXAMPLE 3 Transfection of Pre-Biosensor WIF-1α with pESCleu-RI7-DH5α toForm Biosensor WIF-1α-RI7

Rat olfactory receptor R17, which is responsive to octaldehyde, wascloned by RT-PCR methods using mRNA isolated from rat olfactoryepithelium. This receptor was cloned into modified yeast pESC expressionvector pESCleu-RI7-DH5α, which was transfected into the olfactorypre-biosensor WIF-1α to produce biosensor WIF-1α-R17.

3.1—Construction of pESCleu-RI7-DH5α—The rat olfactory receptor R17receptor was cloned from rat olfactory epithelial lamellae total RNAusing RT-PCR. Reverse transcription of the mRNA into DNA was carried outusing the Ready-To-Go™ T-Primed First-Strand Kit manufactured byAmersham Pharmacia Biotech Inc., USA using the manufacturer's protocol.The following primers were used for the subsequent amplification of theRI7 receptor:

Forward (sense) primer: ATGGAGCGAAGGAACCACAGTGGG (SEQ ID NO: 21) Reverse(antisense) primer: CTAACCAATTTTGCTGCCTTTGTTGG (SEQ ID NO: 22)

The PCR cycling protocol was 1×2 min at 94° C., 30×(30 sec at 94° C., 30sec at 55° C., 1 min at 72° C.), and 1×15 min at 72 ° C. The PCR product(984 bp) was gel-purified and cloned into the cloning vector pCR2.1(Invitrogen). After verifying the insert by sequencing, the insertcontaining RI7 was excised out of pCR2.1 vector with Not I and Sac Irestriction enzymes. The insert was gel-purified and ligated to the NotI-Sac I linearized pESC-LEU vector, and the resultant vector wasverified by sequence analysis. This vector was named pESCleu-RI7-DH5αand propagated in E. coli DH5α.

3.2—Transfection of WIF-1α with pESCleu-RI7-DH5α—To obtain cellscompetent for transfection, several colonies of WIF-1α strain of yeastwere grown 50 ml of YPD medium and incubated at 30° C. for 16-18 hrs.with shaking at 200 rpm to obtain stationary phase cultures (OD₆₀₀greater that 1.5). Thirty ml of the stationary culture was transferredto 300 ml of YPD medium and regrown at 30° C. for 3 hr with shaking (200rpm) until the OD₆₀₀ reached 0.4-0.6. The cells were collected bycentrifugation (1000×g for 5 min at room temperature) and the cellpellet was washed with 25-50 ml of sterile TE by centrifugation.

The competent cells were resuspended in 1.5 ml of freshly preparedsterile TE/LiAc solution (see above). Yeast expression vectorpESCleu-RI7-DH5α (100 ng) and 0.1 mg of carrier DNA (herring testes DNA)in a total volume of 0.015 ml was placed in a 1.5 ml tube into which 0.1ml of competent cells were added and mixed thoroughly. 0.6 ml of sterilePEG/LiAc solution was added and the tube contents mixed by vortexing for10 sec. Following a brief incubation for 30 min at 30° C. in a shakingincubator, 0.07 ml of DMSO was added and gently mixed. The tubes werethen subjected to heat-shock (42° C.) for 15 minutes, after which thetubes were chilled in an ice-water bath for 2 min. The cells werecollected by centrifugation (14000 g for 5 sec at room temperature) andthe cell pellet was resuspended in 0.5 ml of sterile TE buffer. Analiquot (0.1-0.2 ml) of the cells was plated onto a 100-mm petri-dishcontaining SD leu, his, trp, ura drop-out amino acid medium (6.7 g yeastnitrogen base without amino acids, 20 g glucose, 20 g Agar and 1 Literwater containing L-Isoleucine, 30 mg/l; L-Valine, 150 mg/l; L-AdenineHemisulfate, 20 mg/l; L-Arginine, 20 mg/l; L-methionine, 20 mg/l;L-tyrosine, 30 mg/l, L-threonine 200 mg/l; L-Phenylalanine, 50 mg/l, andL-lysine, 30 mg/l, but not L-Leucine, L-Histidine, L-Tryptophan andL-Uracil). This medium selects for yeast cells that harborpESCleu-RI7-DH5α in addition to pESChis-creb-cregfp-DH5α,pESCtrp-Golf-cyc-DH5α and pESCura-β2γ5-DH5α expression vectors.

The petri-dishes were incubated at 30° C. for 24 days or until coloniesappeared. The larger colonies were picked and grown on fresh selectiveSD leu, his, trp, ura drop-out amino acid medium (agar dishes as well asin liquid medium). A yeast biosensor expressing pESChis-creb-cregfp-DH5αin addition to pESCtrp-Golf-cy-DH5α and pESCura-β2γ5-DH5α expressionvectors was thus isolated, and named WIF-1α-R17. WIF-1α-R17 was storedas glycerol stocks (25%) at −80° C.

EXAMPLE 4 Construction of Basic Expression Vector pESCleu-RX-DH5α

Yeast basic expression vector pESCleu-RX-DH5α, containing N- andC-terminal segments from the rat RI7 olfactory receptor flanking acloning cassette, was constructed from pESCleu-RI7-DH5α as describedbelow. This basic expression vector can receive any olfactory receptorhypervariable segment into the cloning cassette and be transfected intoa yeast pre-biosensor cell, where it will express a chimeric olfactoryreceptor.

A cDNA fragment encoding the N-terminal 61 amino acids of RI7 plusunique Bgl II and Pst I cloning sites was generated by PCR frompESCleu-RI7-DH5α. The primers used to generate the fragment were a5′-sense primer that anneals to the segment spanning the Dra III site(nucleotide position 2698 of the pESCleu vector back-bone) ofpESCleu-RI7 (the “5′ Dra III primer”), and a reverse primer (the “NT-RX”primer) that can anneal to nucleotides 183-163 of the RI7-cDNA.

5′ Dra III primer: 5′-AGGGCGATGGCCCACTACGTGAACCATCACCCT-3′ (SEQ ID NO:23) NT-RX primer: 5′-AGAGAGAGATCTGCAGATACATGGGTTTGTGGAGGGT-3′ (SEQ IDNO: 24)

Similarly, a cDNA fragment encoding the C-terminal 35 amino acids of RI7plus unique Bgl II and Spe I cloning sites was generated by PCRamplification from pESCleu-RI7-DH5α using the following primers: a5′-sense primer (the “CT-RX” primer) that can anneal to nucleotides881-898 of the RI7 cDNA, and a reverse primer that can anneal to thesegment spanning the Hind III site (nucleotide position 4140 of thepESCleu vector back-bone) of pESCleu-RI7 (the “3′ Hind III” primer).

CT-RX primer: 5′-AGAGAGAGATCTACTAGTATCTACTGCTTGCGCAAC-3′ (SEQ ID NO: 25)3′ Hind III primer: 5′-CTAGCCGCGGTACCAAGCTTACTCGAGGTCTTC-3′ (SEQ ID NO:26)

The PCR-generated, N-terminus encoding cDNA was cut with Dra III and BglII, whereas the C-terminus encoding fragment was cut with Bgl II andHind III. The fragments were ligated to each other at the Bgl II stickyends to generate a Dra III-Hind III fragment. This fragment containednucleotides 1-183 of the RI7 cDNA (that encode the N-terminal 61 aminoacids of RI7) and nucleotides 881-984 of the RI7 cDNA (that encode theC-terminal 35 amino acids of RI7) flanking a Pst I, Bgl II, and Spe Icloning cassette.

The Dra III-Hind III fragment was cloned into pESC-LEU vector digestedwith Dra III-Hind III to produce pESCleu-RX-DH5α. The sequence of thevector was verified by sequence analysis, and the construct waspropagated in E. coli DH5α.

pESCleu-RX-DH5α was deposited on Feb. 1, 2001 with the patent culturecollection of the National Center for Agricultural Utilization Research,Agricultural Research Service, U.S. Department of Agriculture (NRRL),1815 North University Street, Peoria, Ill. 61604 U.S.A., in accordancewith the Budapest Treaty, and has been assigned accession no. NRRLB-30411.

EXAMPLE 5 Detection of Chemical Agents by WIF-1α-RI7

WIF-1α-R17 was able to detect different concentrations of octaldehyde,as revealed by the expression of GFP seen with a fluorescence reader.Using different alcohols and aldehydes, the specificity of WIF-1α-R17for octaldehyde was also demonstrated.

Pre-biosensor WIF-1α (R17 olfactory receptor negative; see Example 2)and biosensor WIF-1α-R17 (RI7 olfactory receptor positive; see Example3) were grown in SD leu, his, trp, ura amino acid drop-out media (seeExample 3.3, above) for 8 hrs. 3×10⁸ cells were reinoculated in 100 mlof SD dropout media for 16 hrs to induce the expression of the signalingand signal reporter components (Gαolf, β2, γ5, adenylate cyclase, CREB)as well as the RI7 olfactory receptor. At the end of 16 hours, the cellswere seeded into 48- or 96-well plates (7×10⁷ cells/well) and used todetermine the sensitivity and specificity of the WIF-1α-R17 biosensor.

5.1—Sensitivity of WIF-1α-RI7 for Different Olfactants—The yeastpre-biosensor WIF-1α and biosensor WIF-la-RI7 were exposed to each offour different olfactants (hexaldehyde, heptaldehyde, octaldehyde andoctanol) at varying concentrations (25, 50, 75, and 100 nM) for 3 hours,and the GFP fluorescence was measured in a Perkin-Elmer BioAssay 7000plus reader using 485 nm for excitation and 535 nm for emission. Theratio of WIF-1α-RI7 GFP fluorescence values over WIF-1α (control) GFPfluorescence values for each olfactant at each concentration are givenin FIG. 4A. As expected, biosensor WIF-1α-RI7 shows greater sensitivityfor octaldehyde at all concentrations tested.5.2—Specificity of WIF-1α-RI7 for Different Olfactants—The yeastpre-biosensor WIF-1α and biosensor WIF-1α-RI7 were exposed to a singleconcentration (25 nM) of four different olfactants (hexaldehyde,heptaldehyde, octaldehyde and octanol) for 3 hours, and GFP fluorescencewas measured in a Perkin-Elmer BioAssay 7000 plus reader using 485 nmfor excitation and 535 nm for emission. The ratios of WIF-1α-RI7 GFPfluorescence values over WIF-1α (control) GFP fluorescence values foreach olfactant are given in FIG. 4B. As expected, biosensor WIF-1α-RI7exhibited greater specificity for octaldehyde.

EXAMPLE 6 Expression Analyses of Mammalian Olfactory SignalingComponents in WIF-1α-RI7

The expression of mammalian olfactory signaling components was confirmedin yeast biosensor WIF-1α-RI7 as follows.

Detection of RI7 Olfactory Receptor RNA—Total RNA from WIF-1α-RI7 anduntransfected yeast control cells was extracted using the QIAGEN RnaEasykit according to the manufacturer's instructions. The RNA wasreverse-transcribed using the Superscript first strand synthesis system(GIBCO/Invitrogen), and was subjected to PCR amplification with the RI7primers and conditions described in Example 3.1 above. The PCR reactionis expected to amplify a 984 bp RI7 fragment. An aliquot of the PCRreaction was electrophoresed on a 1% agarose gel. FIG. 5A shows that theexpected 984 bp RI7 fragment was present in WIF-1α-RI7 cells, but not incontrol cells.

Detection of CREBP and Gαolf Proteins—Total protein was extracted fromWIF-1-α-RI7 and untransfected yeast control cells using the“bead-bashing” technique described in Dunn B and Wobbe C R (1993),“Preparation of protein extracts from yeast,” in Current Protocols inMolecular Biology, (Ausubel F M et al., eds.), John Wiley & Sons, Inc.,New York, pp. 13.13.1-13.13.9, the disclosure of which is hereinincorporated by reference. The protein extracts were subjected tostandard Western blot analysis using antibodies specific for CREBP (NewEngland Biolabs; Cat. #9192) or the Gαolf protein (Santacruz, Calif.;Cat. #SC-385). FIG. 5B shows that both CREBP and Gαolf proteins werepresent in WIF-1α-RI7, but not in the control cells.

EXAMPLE 7 Membrane Localization of Gαolf in WIF-1α-RI7

To verify the membrane localization of Gαolf in the WIF-1α-RI7biosensor, WIF-1α-RI7 cells expressing Gαolf protein were permeablizedusing 20 units of lyticase (Sigma, Mo.) for 30 min at 30° C.Permeablized cells were collected by centrifugation, and incubated withanti-Gαolf antibody (Santacruz, Calif.; Cat. # SC-385) at 1:200 dilutionfor 1 hr., followed by a further 1 hr. incubation withanti-immunoglobulin antibodies labeled with Texas-red fluorophore(Molecular Probes, Oreg.; Cat # T-2767) at 1:500 dilution. Tenmicroliters of these cells were then mounted on a slide and analyzedwith a laser confocal microscope (Olympus). Red fluorescencerepresenting the Gαolf protein was observed in the WIF-1α-RI7 cells andwas localized to the cell membrane. WIF-1α-RI7 cells stained either withprimary or secondary antibody alone were used as controls, and nosignificant red fluorescence was seen in these cells.

EXAMPLE 8 Detection of GFP Response in WIF-1α-RI7 Cells

WIF-1α-RI7 cells were exposed to 100 nmoles of hexaldehyde (6-CHO),heptaldehyde (7-CHO) or octaldehyde (8-CHO). Control cells were notexposed to any olfactant. At 3 hrs. post-exposure, 10 microliters ofcells were removed from each treatment group and the control group, andmounted separately on slides. GFP expression in the presence of theolfactant was detected in each sample by laser confocal microscopy, andthe results are shown in FIGS. 6A-6D.

FIG. 6A shows the background expression of GFP in the absence ofolfactant. Exposure of the cells to hexaldehyde produced GFP expressionessentially equal to background (see FIG. 6B). FIG. 6C shows anincreased GFP expression in cells exposed to heptaldehyde with respectto the control and hexaldehyde-exposed cells. FIG. 6D shows an increasein GFP expression in cells exposed to octaldehyde with respect toheptaldehyde-exposed cells. These results show that WIF-1α-RI7 cellsspecifically detect octaldehyde, with some detection of heptaldehyde,the closest homolog to octaldehyde. There is no detection ofhexaldehyde, a homolog only two carbons removed from octaldehyde, by theWIF-1α-RI7 cells.

All references discussed herein are incorporated by reference. Oneskilled in the art will readily appreciate that the present invention iswell adapted to carry out the objects and obtain the ends and advantagesmentioned, as well as those inherent therein. The present invention maybe embodied in other specific forms without departing from the spirit oressential attributes thereof and, accordingly, reference should be madeto the appended claims, rather than to the foregoing specification, asindicating the scope of the invention.

1. A biosensor comprising a yeast expressing the following elements: (a)at least one olfactory receptor protein for binding olfactant; (b) atleast one exogenous signaling pathway coupled to the at least oneolfactory receptor for transducing a signal produced by the at least oneolfactory receptor upon olfactant binding; and (c) at least one signalreporter coupled to the signaling pathway for producing a detectablephenomenon upon transduction of the olfactant binding signal by thesignaling pathway,  wherein the at least one olfactory receptor proteincomprises: (i) an olfactory receptor hypervariable segment whichcontains at least one olfactant binding site; (ii) aprocessing/transport segment which directs the processing and transportof the receptor in the yeast, wherein the processing/transport segmentis located N-terminal to the olfactory receptor hypervariable segment;and (iii) a coupling segment which couples the receptor to the at leastone exogenous signaling pathway in the yeast, wherein the couplingsegment is located C-terminal to the olfactory receptor hypervariablesegment.
 2. The biosensor of claim 1, wherein the processing/transportsegment comprises the first 60 amino acids of the yeast mam2 protein,and the coupling segment comprises the last 35 amino acids of the ratRI7 receptor.
 3. The biosensor of claim 1, wherein theprocessing/transport segment comprises the first 61 amino acids of therat RI7 olfactory receptor, and the coupling segment comprises the last35 amino acids of the rat RI7 receptor.
 4. The biosensor of claim 1,wherein the at least one signaling pathway comprises a G-protein and anadenylate cyclase.
 5. The biosensor of claim 1, wherein the at least onesignal reporter is responsive to intracellular cAMP or Ca⁺⁺ levels. 6.The biosensor of claim 5, wherein the at least one signal reporter isselected from the group consisting of a cyclic AMP-responsive GFPexpression system, a cyclic AMP-responsive β-galactosidase expressionsystem, a Ca⁺⁺-responsive luminescence reporter system, a fluorescentcytosolic Ca⁺⁺ indicator, and the electrophysiological detection of Ca⁺⁺influx.
 7. The biosensor of claim 5, wherein the at least one signalreporter comprises GFP.
 8. The biosensor of claim 1, wherein the atleast one signaling pathway comprises an olfactory receptor G-protein,type III adenylyl cyclase, and the at least one signal reportercomprises CREB and CRE-driven GFP.
 9. A method of identifying biosensorswhich can detect a selected olfactant, comprising the steps of: (1)providing at least one biosensor of claim 1; (2) contacting the at leastone biosensor with the selected olfactant; and (3) observing whethersaid detectable phenomenon is produced from the signal reporter of saidbiosensor.
 10. The method of claim 9, wherein the signal is detected byan apparatus comprising a measurement tool for detecting the detectablephenomenon and a computer controller for controlling operation of themeasurement tool.
 11. The method of claim 10, wherein the at least onebiosensor is located in a fixed position on an array comprising a solidsupport.
 12. The method of claim 10, wherein the at least one biosensoris located in a set pattern on a biochip comprising a solid substrate,optionally together with machine readable information encoded on thesubstrate identifying the location and type of the at least onebiosensor.
 13. The method of claim 10, wherein the apparatus isportable.
 14. An apparatus capable of detecting the detectablephenomenon produced by a biosensor of claim 1, comprising a measurementtool for measuring the detectable phenomenon, a computer controller forcontrolling the measurement tool and at least one said biosensor. 15.The apparatus of claim 14, wherein the at least one biosensor is locatedin a fixed position on an array comprising a solid support.
 16. Theapparatus of claim 14, wherein the at least one biosensor is located ina set pattern on a biochip comprising a solid substrate, optionallytogether with machine readable information encoded on the substrateidentifying the location and type of the at least one biosensor.
 17. Amethod for detecting a selected olfactant in a sample, comprising: (1)providing at least one biosensor of claim 1 capable of detecting theolfactant, wherein detection of the olfactant generates a detectablephenomenon from the signal reporter of said biosensor; (2) contactingsaid at least one biosensor with a sample suspected of containing theolfactant; and (3) observing whether said detectable phenomenon isproduced from the signal reporter.
 18. The method of claim 17, whereinsaid detectable phenomenon comprises fluorescence.
 19. The method ofclaim 17, wherein the detectable phenomenon is detected by an apparatuscomprising a measurement tool for detecting the detectable phenomenonand a computer controller for controlling operation of the measurementtool.
 20. The method of claim 19, wherein the at least one biosensor islocated in a fixed position on an array comprising a solid support. 21.The method of claim 19, wherein the at least one biosensor is located ina set pattern on a biochip comprising a solid substrate, optionallytogether with machine readable information encoded on the substrateidentifying the location and type of the at least one biosensor.
 22. Themethod of claim 19, wherein the apparatus is portable.
 23. A library ofbiosensors comprising a plurality of biosensors of claim 1 which expressdifferent olfactory receptor proteins.
 24. An array comprising a solidsubstrate and at least one biosensor of claim 1 arranged in a fixedposition on the substrate.
 25. A biochip comprising a solid substrateand at least one biosensor of claim 1 located in a set pattern on saidsubstrate, optionally together with machine readable information encodedon the substrate identifying the location and type of the at least onebiosensor on the substrate.
 26. A portable container comprising at leastone biosensor of claim
 1. 27. A method of constructing a biosensor,comprising transfecting one or more yeast to express the followingcomponents: (a) at least one olfactory receptor protein for bindingolfactant; (b) at least one exogenous signaling pathway coupled to theat least one olfactory receptor for transducing a signal produced by theat least one olfactory receptor upon olfactant binding; and (c) at leastone signal reporter coupled to the signaling pathway for producing adetectable phenomenon upon transduction of the olfactant binding signalby the signaling pathway,  wherein the at least one olfactory receptorprotein comprises: (i) an olfactory receptor hypervariable segment whichcontains at least one olfactant binding site; (ii) aprocessing/transport segment which directs the processing and transportof the receptor in the host cell, wherein the processing/transportsegment is located N-terminal to the olfactory receptor hypervariablesegment; and (iii) a coupling segment which couples the receptor to theat least one exogenous signaling pathway in the host cell, wherein thecoupling segment is located C-terminal to the olfactory receptorhypervariable segment.
 28. The method of claim 27, wherein theprocessing/transport segment comprises the first 60 amino acids of theyeast mam2 protein, and the coupling segment comprises the last 35 aminoacids of the rat RI7 receptor.
 29. The method of claim 27, wherein theprocessing/transport segment comprises the first 61 amino acids of therat RI7 olfactory receptor, and the coupling segment comprises the last35 amino acids of the rat RI7 receptor.
 30. The method of claim 27,wherein the at least one exogenous signaling pathway comprises aG-protein and an adenylate cyclase.
 31. The method of claim 27, whereinthe at least one signal reporter is responsive to intracellular cAMP orCa⁺⁺ levels.
 32. An expression vector comprising nucleic acid sequencesencoding an olfactory receptor protein, wherein the olfactory receptorprotein comprises a yeast processing/transport segment, which directsthe processing and transport of the receptor in yeast, and a mammaliancoupling segment which couples the receptor to an exogenous signalingpathway in yeast, further wherein the processing/transport segment islocated N-terminal to the olfactory receptor hypervariable segment, andthe coupling segment is located C-terminal to the olfactory receptorhypervariable segment.
 33. An expression vector library comprising aplurality of expression vectors of claim 32, which comprise differentolfactory receptor hypervariable segments.
 34. A method of constructinga library of biosensors, comprising transfecting a plurality ofpre-biosensors with the expression vector library of claim 33 so thatthe pre-biosensors express differing olfactory receptors, wherein eachpre-biosensors comprises: (a) at least one exogenous signaling pathwaycoupled to an olfactory receptor for transducing a signal produced bythe olfactory receptors upon olfactant binding; and (b) at least onesignal reporter coupled to the signaling pathway for producing adetectable phenomenon upon transduction of the olfactant binding signalby the signaling pathway.
 35. An expression vector comprising nucleicacid sequences encoding an olfactory receptor protein, wherein theolfactory receptor protein comprises a processing/transport segmentwhich directs the processing and transport of the receptor in yeast, anda coupling segment which couples the receptor to an exogenous signalingpathway in yeast, further wherein the processing/transport segment islocated N-terminal to the olfactory receptor hypervariable segment, andthe coupling segment is located C-terminal to the olfactory receptorhypervariable segment, further wherein the processing/transport segmentcomprises the first 61 amino acids of the rat RI7 olfactory receptor,and the coupling segment comprises the last 35 amino acids of the ratRI7 receptor.
 36. The expression vector of claim 35, wherein the vectoris pESCleu-RI7-DH5α.
 37. An olfactory receptor protein comprising anolfactory receptor hypervariable segment, a yeast processing/transportsegment, which directs the processing and transport of the receptor inyeast, and a mammalian coupling segment which couples the receptor to anexogenous signaling pathway in yeast, wherein the processing/transportsegment is located N-terminal to the olfactory receptor hypervariablesegment, and the coupling segment is located C-terminal to the olfactoryreceptor hypervariable segment.
 38. A kit comprising: (1) one or morepre-biosensors transfected with at least one exogenous cell signalingpathway; and (2) one or more vectors adapted to receive an olfactoryreceptor hypervariable segment, said one or more vectors comprising acloning cassette and nucleic acid sequences encoding an N-terminalsegment and a C-terminal segment of an olfactory receptor, wherein theN-terminal segment directs the processing and transport of the receptorin yeast, and the C-terminal segment couples the receptor to anexogenous signaling pathway, wherein the vectors further comprisenucleic acid sequences encoding an olfactory receptor proteinhypervariable segment; wherein said pre-biosensor is yeast.